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Article Complete Mitochondrial Genome of trichiura from Macaca sylvanus and Papio papio

Julia Rivero , Rocío Callejón and Cristina Cutillas *

Department of Microbiology and , Faculty of Pharmacy, University of Seville, Calle San Fernando, 4, 41004 Sevilla, Spain; [email protected] (J.R.); [email protected] (R.C.) * Correspondence: [email protected]

Abstract: is among the most prevalent worldwide caused by helminths. For many years, Trichuris spp. have been described with a relatively narrow range of both morphological and biometrical features. The use of the complete mitochondrial genome (mitogenome) is an alternative and powerful molecular method for inferring phylogenies. Here, we present an overview of the contributions of mitogenome for Trichuris spp. from and non-human primates. In addition, we carry out structural and phylogenetic comparative analyses with genomes of Trichuris available in public datasets. The complete mt genomes of and Trichuris sp. from Macaca sylvanus and T. trichiura from Papio papio are 14,091 bp, 14,047 bp and 14,089 bp in length, respectively. The three mt genomes are circular and consist of 37 genes—13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS). The molecular evidence presented here supports the hypothesis that T. trichiura de M. sylvanus (TMF31) and T. trichiura de P. papio (TPM1) were similar but genetically different with respect to Trichuris sp. from macaques (TMM5). The phylogenetic study also supported the evolution of the   different Trichuris species. In conclusion, we suggest the existence of two cryptic species parasitizing M. sylvanus. Citation: Rivero, J.; Callejón, R.; Cutillas, C. Complete Mitochondrial Keywords: complete mitochondrial genome; Trichuris trichiura; phylogeny; whipworms Genome of Trichuris trichiura from Macaca sylvanus and Papio papio. Life 2021, 11, 126. https://doi.org/ 10.3390/life11020126 1. Introduction

Academic Editor: Angela Soil-transmitted helminths (STHs) ( (), Trichuris trichiura Anna Messina (Trichuriasis)) and (/) are widely distributed in Received: 9 January 2021 tropical and subtropical areas, with the greatest numbers occurring in sub-Saharan Africa, Accepted: 3 February 2021 the Americas, China, and East Asia [1]. In areas with favorable climatic and environmental Published: 6 February 2021 conditions, poor access to potable water supply, , and hygiene resources, the transmission of these parasites is higher [2]. Recent data show that about 1.5 billion people Publisher’s Note: MDPI stays neutral are estimated to be affected by STHs worldwide [1]. with regard to jurisdictional claims in Although Trichuriasis is a seriously neglected disease, fortunately, in recent years published maps and institutional affil- hereditary studies are increasing as well as related methodologies that provide new op- iations. portunities for the discovery of novel intervention strategies, with major implications for improving and human health and welfare globally [3]. In addition, the implications of genomic studies could also be very relevant in relation to the search for new treatments for immunopathological diseases in [4–9]. For example, it has been reported that Copyright: © 2021 by the authors. infections of human patients suffering from immunological disorders (such as Crohn’s Licensee MDPI, Basel, Switzerland. disease) suppress clinical symptoms significantly with pig-Trichuris eggs [6,10–12]. This article is an open access article The mitogenome (complete mitochondrial genome) has several practical distributed under the terms and strengths as a phylogenetic marker, and has yielded well-supported results for clades, conditions of the Creative Commons which were not well resolved using other approaches [13]. The whole genome and tran- Attribution (CC BY) license (https:// scriptome studies have contributed to our understanding of deep node nematode phy- creativecommons.org/licenses/by/ logeny but have been limited by low taxon sampling (which is also biased toward some 4.0/).

Life 2021, 11, 126. https://doi.org/10.3390/life11020126 https://www.mdpi.com/journal/life Life 2021, 11, 126 2 of 15

parasitic groups) and by the technical challenges inherent in obtaining a large quantity of genetic information from a single nematode with a small body size. In such cases, the use of mitogenome sequences is one alternative that has been widely applied to many nematode branches where relationships were unclear [13]. Nematode mitogenomes are like those of other in many respects, but have a few unusual features including high variation in conservation of gene across ma- jor branches and the occasional presence of multiple chromosomes [13]. The nematode mitogenome is usually a single, circular molecule ranging in size from 12 to 22 kb and con- taining 36 (sometimes 37) genes—12 (or 13) protein coding genes (PCGs) (cox1–cox3, cytb, nad1-nad6, nad4L, atp6, and rarely atp8), two ribosomal RNA (rRNA) (rrnL and rrnS), and 22 transfer RNA genes (tRNAs). The atp8 gene, which is found in most other metazoan mi- togenomes (except the parasitic Platyhelminthes clade Neodermata) [14], is usually absent in nematode mitogenomes, although it does appear in the order Trichinellida ( spp. and Trichuris spp.) [13,15–20]. Furthermore, within , members of order Trichinellida (Trichinella spp., Trichuris spp.) show a substantial gene rearrangement even among closely related species, while members of show far less rearrangement in theirs mitogenomes [21]. Kern et al. [13] concluded that the mitochondrial genome is a useful tool for nematode phylogenetic because the diversity within nematode mitogenome architecture, its variable rate of gene rearrangement, and the representation of nearly every kind of lifestyle and habitat ecology within make this an exciting area for addressing questions about mitogenome evolution. Currently, the complete mt genome of several species of whipworms has been sequenced— Trichuris trichiura from humans and baboons [3,19]; Trichuris sp. from the Endangered François’ Leaf-Monkey [17]; Trichuris rhinopiptheroxella from the endangered golden snub-nosed monkey [20]; from pig [3,19]; from ante- lope [16]; from wild yak [16]; Trichuris skrjabini from sheep [22]; and (LC050561, unpublished). This fact, and considering the hypothesis that Trichuris infecting primates represent a complex of cryptic species with some species capa- ble of infecting both humans and non-human primates (NHPs), reflects the need to delve into the mt genomes of Trichuris spp. of NHPs compared to T. trichiura from humans. Thus, the aim of the present study was to sequence the complete mt genome of Trichuris trichiura isolated from different NHPs. In addition, we carry out structural and phylogenetic comparative analyses with genomes of species of Trichuris available in public datasets.

2. Materials and Methods 2.1. Ethics Statement This study does not require approval by an ethics committee. Macaca sylvanus, from which Trichuris specimens were collected from their caeca post-mortem. Trichuris specimens, recovered from the feces of one Papio papio after treatment, were handled and housed in a zoo in strict accordance with good animal practices.

2.2. Parasites, DNA Extraction and Genotyping of Worms Specimen Trichuris worms were recollected from a Guinea baboon (P. papio) at Parque de la Naturaleza de Cabárceno (Cantabria, Spain), from a stool sample after anthelmintic treatment, and a Barbary macaque (M. sylvanus) in Castellar Zoo (Cádiz, Spain), from its caeca post-mortem. Adult Trichuris were recovered and washed in physiological saline, identified morphologically, and then, stored at −20 ◦C until use. Total genomic DNA was isolated from three individual worms according to the manufacturer’s protocol the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Düsseldorf, Germany),used to extract the genomic DNA. Quality of extractions was assessed using 0.8% agarose gel electrophoresis infused with SYBR® Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA, USA) Life 2021, 11, 126 3 of 15

2.3. Mitochondrial Genome Amplification and Sequencing For long-range PCR amplification and next generation sequencing (NGS), different primate derived Trichuris specimens were chosen. One Guinea baboon worm (TPM1) and two Barbary macaque worms (TMF31 and TMM5) were chosen based on their distinct haplotypes identified in previous studies by sequencing the partial gene of mtDNA (cox1, cob and rrnL) and rDNA (ITS1 and ITS2) [23]. To obtain the complete mt genome, firstly, we used the primers designed by Hawash et al. [19] to amplify the Trichuris sp. from baboon (TTB1) and T. trichiura from humans from Uganda (TTHUG) genomes in two overlapping fragments (~8 and ~6 kbp), Nevertheless, we only could amplify the second fragment (~6 kbp), from rrnL to nad1. Then, new primers (MS1F and MS1R) were designed to amplify the other fragment using Primer3 from nad1 to rrnL (http://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 1 February 2021)). PCR mix, PCR conditions and PCR primers are summarized in the Supplementary materials (Table S1). The PCR products were checked on SYBR® Safe stained 0.8% Tris-Borate-EDTA (TBE) agarose gels and bands were eluted and purified from the agarose gel using the QWizard SV Gel and PCR Clean Up System Kit (Promega, Madison, WI, USA). Once purified, the samples were concentrated and measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Stab Vida (Lisbon, Portugal) sequenced the mt genomes. The PCR products were used for library construction using Illumina Nextera XT library preparation kit (San Diego, CA, USA) and the generated DNA fragments were sequenced in the Illumina MiSeq platform (San Diego, CA, USA), using 300 bp paired-end sequencing reads.

2.4. Assembly, Annotation, and Genome Sequence Analyses Sequences were assembled and analyzed using MacVector package v17.5.4 (Oxford Molecular Group, Waterbeach, Cambridge, UK). The identity of the sequences was made using BLAST by comparison with other sequences available in GenBank database. Genome annotation was performed using the pipeline MITOS web server (http://mitos.bioinf.uni- leipzig.de/index.py (accessed on 1 February 2021)) [24] using the mitochondrial inverte- brate genetic code, and MacVector and protein-encoding genes of T. trichiura. The majority of the tRNA genes were identified using the ARWEN tool (http://130.235.244.92/ARWEN/ (accessed on 1 February 2021)) to get rRNA [25] and tRNAScan-SE web server (http: //trna.ucsc.edu/tRNAscan-SE/ (accessed on 1 February 2021)) [26], but the remaining tRNA genes were recognized manually by comparison. The genomes were compared with T. trichiura and Trichuris sp. Sequences—T. trichiura from humans (unknown geographical location) (AP017704), T. trichiura from humans in China (NC_017750), T. trichiura from humans in China (GU385218), T. trichiura from humans in Uganda (KT449826), T. trichiura from P. anubis in USA (KT449825), T. trichiura from P. hamadryas in Denmark (KT449824) and Trichuris sp. from T. francoisi in China (KC461179). All protein-coding genes (PCGs) and ribosomal RNA genes (rRNAs) sequences were individually extracted, and a nucleotide data set was generated by concatenated sequences (all PCGs and rRNAs genes). This data set was used to estimate genetic distances using MEGA X v.10.1.8 (Penn State, PA, USA) [27]. The genomes of Trichuris from humans and NHP were used to calculate the < Nucleotide diversity (π) using a sliding window of 100 bp with 25 bp steps implemented in DnaSP v.6 [28].

2.5. Phylogenetic Analysis For the phylogenetic analyses, two different data sets were generated. The first included nucleotides sequences, with the 13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cob) and the two rRNAs (rrnL and rrnS). The data set was aligned using MEGA X v.10.1.8 [27] and concatenated for Trichuris sp. and T. trichiura species from baboons and humans. We used as an outgroup Trichinella pseudospiralis (Table1). Life 2021, 11, 126 4 of 15

Table 1. Sequences analyzed in the first data set for phylogenetic analyses.

Species Host Species/Geographical Origin GenBank Accession Number Trichuris trichiura Macaca sylvanus/Spain MW448470 Trichuris sp. Macaca sylvanus/Spain MW448471 Trichuris trichiura Papio papio/Spain MW448472 Homo sapiens/(unknown Trichuris trichiura AP017704 geographical location) Trichuris trichiura Homo sapiens/China NC_017750 Trichuris trichiura Homo sapiens/China GU385218 Trichuris trichiura Homo sapiens/Uganda KT449826 Trichuris sp. Papio anubis/USA KT449825 Trichuris sp. Papio hamadryas/Denmark KT449824 Trichuris sp. Trachypithecus francoisi/China KC461179 1Trichinella pseudospiralis Coragypus atratus/USA KM357411 1 Used as an outgroup.

Another data set was generated using amino acid sequences inferred from the 12 PCGs. The sequences were aligned using MEGA X v.10.1.8 [27] and then concatenated excluding atp8 (because it is not present in the mitochondrial genome of all the species of nematodes except in Trichinella and Trichuris species). The data set, using the sequences previously used and all the sequences available of the complete mt genome of Trichuris spp. and with those of 9 other enoplid nematodes, using and Ascaris suum as the outgroups, was generated (Table2). The ambiguous regions of the alignment were excluded using Gblocks Server v.0.91b (http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks (accessed on 1 February 2021)) with the default settings being used to select the option of less strict conservation of flanking positions [29,30].

Table 2. Sequences analyzed in the second data set for phylogenetic analyses.

Host Species/ GenBank Species Order Geographical Origin Accession Number Trichuris trichiura Macaca sylvanus/Spain Trichinellida MW448470 Trichuris sp. Macaca sylvanus/Spain Trichinellida MW448471 Trichuris trichiura Papio papio/Spain Trichinellida MW448472 Homo sapiens/(unknown Trichuris trichiura Trichinellida AP017704 geographical location) Trichuris trichiura Homo sapiens/China Trichinellida NC_017750 Trichuris trichiura Homo sapiens/China Trichinellida GU385218 Trichuris trichiura Homo sapiens/Uganda Trichinellida KT449826 Trichuris sp. Papio anubis/USA Trichinellida KT449825 Trichuris sp. Papio hamadryas/Denmark Trichinellida KT449824 Trichuris sp. Trachypithecus francoisi/China Trichinellida KC461179 Trichuris Rhinopithecus roxellana/China Trichinellida MG189593 rhinopiptheroxella Trichuris ovis Addax nasomaculatus/China Trichinellida NC_018597 Trichuris discolor Bos grunniens mutus/China Trichinellida NC_018596 Trichuris muris - /United Kingdom Trichinellida LC050561 Trichuris suis Sus scrofa/Uganda Trichinellida KT449823 Trichuris suis Sus scrofa/China Trichinellida GU070737 Trichinella Coragypus atratus/USA Trichinellida KM357411 pseudospiralis Xiphinema americanum Plant ectoparasite NC_005928 Hexamermis agrotis - NC_008828 Agamermis sp. - Mermithida NC_008231 Romanomermis - Mermithida NC_008640 culicivorax Life 2021, 11, 126 5 of 15

Table 2. Cont.

Host Species/Geographical GenBank Accession Species Order Origin Number Romanomermis - Mermithida NC_008693 iyengari Romanomermis nielseni - Mermithida NC_008692 Strelkovimermis - Mermithida NC_008047 spiculatus Thaumamermis - Mermithida NC_008046 cosgrovei 1Brugia malayi - NC_004298 1Ascaris suum - Rhabditida HQ704901 1 Used as an outgroup.

For phylogenetic re-constructions we used three methods—Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inferences (BI). The ML tree was generated using PHYML package [31,32], and for the MP tree we used MEGA X v.10.1.8 [27] and for BI we used MrBayes v.3.2.6. [33]. To resolve the best-fit substitution model for the nucleotide data set we employed jModelTest [34] and ProtTest 3.4 for the amino acid data set. Models of evolution were defined according to the Akaike Information Criterion [35,36]. For the nucleotide data set, GTR + I + G model, with rate variation along the length of the alignment (+ G) and allowing for a proportion of invariant sites (+ I) was selected, and for the amino acid data set, the MtArt + I + G + F model, with residue frequencies estimated from the data (+ F) was chosen. Support for the topology was examined using bootstrapping (heuristic option) [37] over 1000 replications to assess the relative reliability of clades. The commands used in MrBayes for BI were nst = mixed. The standard deviation of split frequencies was used to determine whether the number of generations completed was sufficient; the chain was sampled every 500 generations and each dataset was run for 10 million generations. Trees from the first million generations were discarded based on an assessment of convergence. Burn-in was determined empirically by examination of the log likelihood values of the chains. The Bayesian posterior probabilities (BPPs) comprise the percentage converted.

3. Results 3.1. Annotation and Features of Mitochondrial Genomes The complete mtDNA sequences of the primate worms TMF31, TMM5 and TPM1 were 14,091, 14,047 and 14,089 bp in length, respectively (GenBank accession nos. MW448470-2) (Figure1). The mt genomes contained 37 genes—13 PCGs ( cox1–3, nad1–6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS) (Table3). All genes are transcribed from the heavy strand, except four PCGs (nad2, nad4, nad4L and nad5) and 10 tRNA (tRNA-Met, tRNA-Phe, tRNA-His, tRNA-Arg, tRNA-Pro, tRNA-Trp, tRNA-Ile, tRNA-Gly, tRNA-Cys, and tRNA-Tyr) that are transcribed from the light strand. The mt genomes of Trichuris sp. contain an AT-rich region consisting of two non- coding regions (NCRs), including a long non-coding region (NCR-L) and short non-coding region (NCR-S). The nucleotide composition (%) is summarized in Table S3. The content of A + T is 69.4%, 68% and 69.3% for TMH31, TMM5 and TPM1, respectively. Between TMF31 and TPM1 genomes there were slight differences. The sequences were identical in terms of all initiation and termination codons and gene lengths, except for tRNA-ser (S2), which presented one nucleotide more in TPM1. In addition, between TMF31 and TMM5, 22 genes of 37 were different in length. LifeLife 20212021,, 1111,, x 126 FOR PEER REVIEW 6 6of of 16 15

FigureFigure 1. MitochondrialMitochondrial genome genome structure structure of of TrichurisTrichuris trichiura fromfrom Barbary Barbary macaque macaque (TMF31) (TMF31) and and from from Guinea Guinea baboon baboon (TPM1),(TPM1), and and Trichuris sp. from Barbary macaque (TMM5). Genes Genes were were re representedpresented to to standard standard nomenclature, nomenclature, but but tRNAs tRNAs were represented using one-letter amino amino ac acidid codes, codes, with with numbers numbers differentiating differentiating each each of of the the two two leucine- leucine- and and serine- serine- specifyingspecifying tRNAs. tRNAs. NCR-L NCR-L refers refers to to large large non-codi non-codingng region and NCR-S to small non-coding region.region.

Table 3. Mitochondrial genomes Theof Trichuris start/stop from Macaca codons sylvanus for some (TMF31, PCGs TMM5) differed andbetween Papio papio TMM5 (TPM1). and Protein the genomescod- ing, transfer RNA (tRNA), andpreviously ribosomal cited RNA (TMF31 (rRNA) genes and TPM1), with lengths which in werenucleotides similar. (nt) Codonare given. usage The lengths analyses are of mt not identical, and differencesgenomes are given showedin parentheses that three (TMM start5/TPM1), and sevenlikewise termination for the initiation codons and weretermination different codons. (Table 3). ForPositions instance, the starting codon forLengths TMF31 and TPM1 is ATACodons for the nad1 gene, while it Genes reads ATG in the TMM5 genome; for the nad4 gene, the starting codon is ATG inStrand TMF31 TMF31 TMM5 TPM1 nt Initiation Termination and TPM1, while being ATA in TMM5, and in the atp8 gene, ATT is the start codon in cox1 1–1545 1–1545 1–1545 1545 ATG TAA (TAG) + TMF31 and TPM1, while it reads ATA in TMM5. In the cox1 gene, TAA is the termination cox2 1558–2232 1556–2230 1558–2232 675 ATG TAG (TAA) + codon in TMF31 and TPM1, whereas it is TAG in TMM5; in cox2 gene, TAG is the stop tRNA-leu (L2) 2255–2317 2252–2311 2255–2317 63 (60) + codon in TMF31 and TPM1, while being TAA in TMM5; in nad1, TAG is the stop codon in tRNA-glu (E) 2324–2384 2320–2377 2324–2384 61 (58) + TMF31 and TPM1, but is TAA in TMM5; in nad2 gene, TAA is the stop codon in TMF31 and nad1 2406–3305 TPM1, 2401–3300 and is TAG 2406–3305 in TMM5; the stop 900 codon in the ATAnad (ATG)5 gene is TAG TAG for (TAA) TMF31 and + TPM1 Non-coding re- 3306–3435 and3303–3442 TAA for TMM5; 3306–3430 in the nad4L and nad6 genes, TAA is the stop codon in TMF31 and gion (NCR-L) TPM1 while it reads TAG in TMM5, and in cox3 gene TAA is the stop codon for TMF31 and tRNA-lys (K) 3436–3501 3441–3502 3431–3496 66 (62) +

Life 2021, 11, 126 7 of 15

TPM1 and TAG for TMM5. There are overlaps between rrnL and atp6 in TMF31, between rrnL, atp6 and cox3 in TMM5, and between rrnL and atp6 in TPM1.

Table 3. Mitochondrial genomes of Trichuris from Macaca sylvanus (TMF31, TMM5) and Papio papio (TPM1). Protein coding, transfer RNA (tRNA), and ribosomal RNA (rRNA) genes with lengths in nucleotides (nt) are given. The lengths are not identical, and differences are given in parentheses (TMM5/TPM1), likewise for the initiation and termination codons.

Positions Lengths Codons Genes TMF31 TMM5 TPM1 nt Initiation Termination Strand cox1 1–1545 1–1545 1–1545 1545 ATG TAA (TAG) + cox2 1558–2232 1556–2230 1558–2232 675 ATG TAG (TAA) + tRNA-leu (L2) 2255–2317 2252–2311 2255–2317 63 (60) + tRNA-glu (E) 2324–2384 2320–2377 2324–2384 61 (58) + nad1 2406–3305 2401–3300 2406–3305 900 ATA (ATG) TAG (TAA) + Non-coding region (NCR-L) 3306–3435 3303–3442 3306–3430 tRNA-lys (K) 3436–3501 3441–3502 3431–3496 66 (62) + nad2 3499–4395 3505–4401 3494–4390 897 ATA TAA (TAG) − tRNA-met (M) 4396–4456 4402–4462 4391–4451 61 − tRNA-phe (F) 4451–4507 4457–4513 4446–4502 57 − nad5 4499–6055 4520–6067 4494–6050 1557 (1553) ATA TAG (TAA) − tRNA-his (H) 6049–6106 6065–6118 6044–6101 58 (54) − tRNA-arg (R) 6108–6171 6115–6181 6103–6166 64 (67) − nad4 6176–7396 6183–7394 6171–7391 1221 (1212) ATG (ATA) TAA − nad4L 7419–7631 7425–7673 7414–7626 213 (249) ATA TAA (TAG) − tRNA-thr (T) 7672–7729 7679–7736 7667–7724 58 + tRNA-pro (P) 7729–7787 7736–7795 7724–7782 59 (60) − nad6 7780–8256 7788–8264 7775–8251 477 ATT TAA (TAG) + cob 8263–9369 8272–9378 8258–9364 1107 ATG TAG + tRNA-ser (S1) 9368–9420 9377–9426 9363–9415 53 (50) + rrnS 9413–10116 9419–10112 9408–10,111 704 (694) + tRNA-val (V) 10,118–10,174 10,114–10,170 10,113–10,169 57 + rrnL 10,176–11,184 10,170–11,180 10,171–11,179 1009 (1011) + atp6 11,155–11,967 11,151–11,990 11,150–11,962 813 (840) ATG TAA + cox3 11,973–12,746 11,965–12,738 11,968–12,741 774 ATG TAA (TAG) + tRNA-trp (W) 12,759–12,821 12,743–12,805 12,754–12,816 63 − tRNA-gln (Q) 12,825–12,880 12,807–12,862 12,820–12,875 56 + tRNA-Ile (I) 12,883–12,943 12,864–12,925 12,878–12,938 61 (62) − tRNA-gly (G) 12,957–13,013 12,934–12,989 12,952–13,008 57 (56) − tRNA-asp (D) 13,020–13,077 12,996–13,060 13,015–13,072 58 (65) + atp8 13,066–13,233 13,042–13,209 13,061–13,228 168 ATT (ATA) TAG + nad3 13,243–13,584 13,219–13,560 13,238–13,579 342 ATT TAA + Non-coding region NCR-S) 13,585–13,676 13,561–13,659 13,580–13,672 tRNA-ser (S2) 13,677–13,726 13,660–13,709 13,673–13,723 50 (50/51) + tRNA-asn (N) 13,727–13,781 13,710–13,763 13,724–13,778 55 (54) + tRNA-leu (L1) 13,789–13,848 13,770–13,835 13,786–13,845 60 (66) + tRNA-ala (A) 13,860–13,917 13,842–13,897 13,857–13,914 58 (56) + tRNA-cys (C) 13,960–14,013 13,924–13,976 13,957–14,010 54 (53) − tRNA-tyr (Y) 14,014–14,074 13,977–14,038 14,011–14,071 61 (62) − Total length 14,091 14,047 14,089

3.2. Comparative Sequence Analyses Genetic distances between worms for individual PCGs and rRNAs genes are found in Table S2. The genetic distances between the mt genomes of Trichuris spp. in primates and humans are given in Table4. The gene with highest genetic variation was the atp8 gene, and the most conserved was the rrnS gene, however between all PCGs, cox1 was the most conserved gene. Among the mt genomes obtained in this study, nucleotide and amino acid differences between TMF31 and TPM1 were 0.25% and 0.41%, and between TMF31 and TMM5 they were 18.7% and 14.5%, respectively. Within all sequences of Trichuris spp. studied, the mt genome of T. francoisi was the most variable (with a nucleotide difference of 27.1–28.6% and an amino acid difference of 26.8–28.2%) relative to the other worms from baboons and humans. Life 2021, 11, 126 8 of 15

Table 4. Pairwise genetic and protein distances between the different complete mt genomes for different Trichuris trichiura and Trichuris sp. in different primates and human hosts at different countries. The nucleotide distances are given below the diagonal and the amino acid distances above the diagonal.

KT2449826 T. KT449825 KT449824 AP017704 T. trichiura H. NC_017750 T. GU385218 T. KC461179 trichiura H. Trichuris sp. Trichuris sp. P. TMF31 TPM1 TMM5 sapiens (Unknown trichiura H. trichiura H. Trichuris sp. GHL sapiens TTB2 P. hamadryas Geographical Location) sapiens China sapiens China T. francoisi China Uganda anubis USA Denmark TMF31 0.41 14.5 14.7 14.8 14.8 0.6 14.6 0.49 26.9 TPM1 0.25 14.6 14.8 14.9 14.9 0.7 14.6 0.34 26.8 TMM5 18.7 18.7 10.9 11.1 11.1 14.5 10.3 14.6 28.2 AP017704 T. trichiura H. sapiens (unknown 18.7 18.7 15.7 4.68 4.68 14.7 3.47 14.9 28.3 geographical location) NC_017750 T. trichiura H. 18.6 18.6 15.9 6.51 0 14.8 4.66 14.9 27.8 sapiens China GU385218 T. trichiura H. 18.6 18.6 15.9 6.51 0 14.8 4.66 14.9 27.8 sapiens China KT2449826 T. trichiura H. 0.4 0.47 18.8 18.7 18.6 18.6 14.5 0.78 26.8 sapiens Uganda KT449825 Trichuris sp. 18.8 18.8 15.8 4.7 6.48 6.48 18.8 14.7 28.2 TTB2 P. anubis USA KT449824 Trichuris sp. P. 0.34 0.28 18.8 18.7 18.6 18.6 0.55 18.8 26.8 hamadryas Denmark KC461179 Trichuris sp. 27.1 27.1 28.4 28.3 28 28 27.1 28.6 27.2 GHL T. francoisi China Life 2021, 11, x FOR PEER REVIEW 8 of 16

and is TAG in TMM5; the stop codon in the nad5 gene is TAG for TMF31 and TPM1 and TAA for TMM5; in the nad4L and nad6 genes, TAA is the stop codon in TMF31 and TPM1 while it reads TAG in TMM5, and in cox3 gene TAA is the stop codon for TMF31 and TPM1 and TAG for TMM5. There are overlaps between rrnL and atp6 in TMF31, between rrnL, atp6 and cox3 in TMM5, and between rrnL and atp6 in TPM1. 3.2. Comparative Sequence Analyses Genetic distances between worms for individual PCGs and rRNAs genes are found in Table S2. The genetic distances between the mt genomes of Trichuris spp. in primates and humans are given in Table 4. The gene with highest genetic variation was the atp8 gene, and the most conserved was the rrnS gene, however between all PCGs, cox1 was the most conserved gene. Among the mt genomes obtained in this study, nucleotide and amino acid differences between TMF31 and TPM1 were 0.25% and 0.41%, and between TMF31 and TMM5 they were 18.7% and 14.5%, respectively. Within all sequences of Tri- churis spp. studied, the mt genome of T. francoisi was the most variable (with a nucleotide Life 2021, 11, 126 difference of 27.1–28.6% and an amino acid difference of 26.8–28.2%) relative to9 ofthe 15 other worms from baboons and humans. Among the Trichuris genome dataset, the nucleotide diversity was analyzed using the slidingAmong window the Trichuris approachgenome for the dataset, 13 PCGs the nucleotide and the two diversity rRNA was genes. analyzed The usingnumber of the sliding window approach for the 13 PCGs and the two rRNA genes. The number of polymorphic sites was 5108 and the nucleotide diversity was 0.166 (Figure 2). The genes polymorphic sites was 5108 and the nucleotide diversity was 0.166 (Figure2). The genes withwith lowest lowest nucleotide nucleotide diversity diversity werewere rRNAs rRNAs (rrn (rrnL andL andrrn rrnS) andS) andcox1. cox1.

Figure 2. Nucleotide diversity (π) for all protein-coding genes (PCGs) and ribosomal rRNA (rrnS and rrnL) measured using Figurea sliding2. Nucleotide window ofdiversity 100 bp with (π) 25for bp all steps. protein-coding The aligned datasetgenes for(PCGs)Trichuris andin ribosomal primates (baboons, rRNA (rrn humansS and and rrn Francois’L) measured usingleaf a sliding monkey). window of 100 bp with 25 bp steps. The aligned dataset for Trichuris in primates (baboons, humans and Francois’ leaf monkey). 3.3. Phylogenetic Analyses The phylogenetics analyses of nucleotide sequence datasets with partial genome (the 13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cytb) and the two rRNAs (rrnL and rrnS)) and with mt genome complete datasets reflecting similar tree topologies by the three methods studied (ML, MP and BI) (Figure3). Within Trichuris populations from humans and NHPs, Trichuris sp. from T. francoisi appeared separated of the other sequences. Within this last group, there are three main clades. The first clade (clade 1) is composed of sequences of Trichuris sp. from H. sapiens and P. anubis from Asia, Japan and USA, the clade 2 is composed of a sequence of Trichuris sp. from M. sylvanus, and the clade 3 corresponded with T. trichiura from humans and NHPs (M. sylvanus, P. papio and P. hamadryas) from Europe and Africa. Phylogenetic trees inferred from a concatenated amino acid sequence dataset of 12 PCGs among selected enoplid nematodes, using the chromadorean nematode, B. malayi (NC_004298) and A. suum (HQ704901) as outgroups, revealed congruence with phylo- genetics analyses of nucleotide sequence datasets with the 13 PCGs and the two rRNAs (rrnL and rrnS), and complete mt genomes. Thus, phylogenetics analyses of all 12 genes showed a general pattern with very strong support for clades near the terminals of the tree (Figure4) . Thus, clade 1, clade 2 and clade 3 for Trichuris spp. from humans and NHPs, and the monophyly of the genus Trichuris, are very strongly supported (100% ML BV, 100% MP BV and 100% BPP). There are no marked differences in support values between the 12 gene datasets analyzed using nucleotide versus amino acid sequences. Life 2021, 11, x FOR PEER REVIEW 10 of 16

3.3. Phylogenetic Analyses The phylogenetics analyses of nucleotide sequence datasets with partial genome (the 13 PCGs (cox1-3, nad1-6, nad4L, atp6, atp8 and cytb) and the two rRNAs (rrnL and rrnS)) and with mt genome complete datasets reflecting similar tree topologies by the three methods studied (ML, MP and BI) (Figure 3). Within Trichuris populations from humans and NHPs, Trichuris sp. from T. francoisi appeared separated of the other sequences. Within this last group, there are three main clades. The first clade (clade 1) is composed of sequences of Trichuris sp. from H. sapiens and P. anubis from Asia, Japan and USA, the clade 2 is composed of a sequence of Trichuris sp. from M. sylvanus, and the clade 3 corre- Life 2021, 11, 126 sponded with T. trichiura from humans and NHPs (M. sylvanus, P. papio and P. hamadryas) 10 of 15 from Europe and Africa.

Life 2021, 11, x FOR PEER REVIEW 11 of 16

BV and 100% BPP). There are no marked differences in support values between the 12 gene datasets analyzed using nucleotide versus amino acid sequences. The phylogenetic analyses reflected the clear distinctiveness between T. trichiura and Figure 3. Phylogenetic tree basedTrichuris on concatenated sp. from humans nucleotide and NHPs sequences with respect of 13 PCGs to T. andsuis twofrom rRNA suids, of T.Trichuris ovis and from T. discolor from herbivorous and T. muris from rodents and grouping these members of Tri- baboons, humansFigure and 3. Phylogenetic françois’ leaf tree monkey based on usingconcatenatedTrichinella nucleo pseudospiralistide sequences ofas 13 an PCGs outgroup, and two inferredrRNA of Trichuris using Bayesian from ba- Inference boons, humans and françois’churis withleaf monkey T. pseudospiralis using Trichinella (order pseudospiralis Trichinellida) as an outgroup, with inferredabsolute using support Bayesian (100% Inference ML BV, MP (BI). Maximum Likelihood (ML) bootstrap values of clades are listed first, followed by Maximum Parsimony (MP) and by (BI). Maximum LikelihoodBV 100% (ML) bootstrapand 100% values BPP), of clades but areexcluding listed first, thefollowed members by Maximum of Dorylaimida Parsimony (MP) and and Mermithida by Bayesian PosteriorBayesian Probabilities Posterior Probabilities(Figure (BPP), 4). for (BPP), clade for clade frequencies frequencies exceeding exceeding 60%. Phylogenetic trees inferred from a concatenated amino acid sequence dataset of 12 PCGs among selected enoplid nematodes, using the chromadorean nematode, B. malayi (NC_004298) and A. suum (HQ704901) as outgroups, revealed congruence with phyloge- netics analyses of nucleotide sequence datasets with the 13 PCGs and the two rRNAs (rrnL and rrnS), and complete mt genomes. Thus, phylogenetics analyses of all 12 genes showed a general pattern with very strong support for clades near the terminals of the tree (Figure 4). Thus, clade 1, clade 2 and clade 3 for Trichuris spp. from humans and NHPs, and the monophyly of the genus Trichuris, are very strongly supported (100% ML BV, 100% MP

FigureFigure 4. Phylogenetic 4. Phylogenetic tree tree among among enoplid enoplid nematodes nematodes based based on on concatenated concatenated amino acid acid sequences sequences of of 12 12 PCGs PCGs (except (except for atp8 gene)for atp by8 gene) Bayesian by Bayesian Inference Inference (BI) using (BI) usingBrugia Brugia malayi malayiand Ascarisand Ascaris suum suumas theas the outgroups. outgroups. Maximum Maximum LikelihoodLikelihood (ML) bootstrap(ML) values bootstrap of cladesvalues areof clades listed are first, listed followed first, followed by Maximum by Maximum Parsimony Parsimony (MP) (MP) and and Bayesian Bayesian Posterior Posterior Probabilities, Probabili- for clade frequenciesties, for clade exceedingfrequencies 60%. exceeding 60%.

4. Discussion Previous studies have reported the hypothesis that a complex whipworm species ex- ists in primates, suggesting that different Trichuris species infect primates and humans [3,19,23,38–41]. In the present study, we resolved the complete mt genome sequences of two haplo- types of Trichuris isolated from M. sylvanus and one complete mt genome of Trichuris from P. papio. The size of the complete mt genomes were within the range reported for the Tri- churidae family (ranging from 13,904 bp (T. discolor) to 14,521 bp (T. suis)) [3,16,17,19,22]. The complete mtDNA is a circular molecule and encodes 37 genes—13 PCGs (atp6, atp8, cox1-3, cob, nad1-6 and nad4L), two rRNAs (rrnS and rrnL), and 22 for tRNAs. The initiation codons (ATG, ATA and ATT) agreed with those reported in the pig- derived and human-derived Trichuris. [16,18,20], while T. ovis and T. discolor also em- ployed TTG [3,16,19], and T. skrjabini sequence used only two start codons (ATG and

Life 2021, 11, 126 11 of 15

The phylogenetic analyses reflected the clear distinctiveness between T. trichiura and Trichuris sp. from humans and NHPs with respect to T. suis from suids, T. ovis and T. discolor from herbivorous and T. muris from rodents and grouping these members of Trichuris with T. pseudospiralis (order Trichinellida) with absolute support (100% ML BV, MP BV 100% and 100% BPP), but excluding the members of Dorylaimida and Mermithida (Figure4).

4. Discussion Previous studies have reported the hypothesis that a complex whipworm species exists in primates, suggesting that different Trichuris species infect primates and hu- mans [3,19,23,38–41]. In the present study, we resolved the complete mt genome sequences of two hap- lotypes of Trichuris isolated from M. sylvanus and one complete mt genome of Trichuris from P. papio. The size of the complete mt genomes were within the range reported for the family (ranging from 13,904 bp (T. discolor) to 14,521 bp (T. suis)) [3,16,17,19,22]. The complete mtDNA is a circular molecule and encodes 37 genes—13 PCGs (atp6, atp8, cox1–3, cob, nad1–6 and nad4L), two rRNAs (rrnS and rrnL), and 22 for tRNAs. The initiation codons (ATG, ATA and ATT) agreed with those reported in the pig- derived and human-derived Trichuris.[16,18,20], while T. ovis and T. discolor also employed TTG [3,16,19], and T. skrjabini sequence used only two start codons (ATG and ATA) [22]. Two termination codons (TAA and TAG) were used as stop codons in all the four species of Trichuridae family (T. suis, T. trichiura, T. ovis and T. discolor) except T. skrjabini, which possesses another stop codon, TGA [3,16,17,19,22]. The sequences of this study were in accordance with the most related species (primates derived Trichuris)[3,19]. The complete mt genomes of one of the haplotypes of Trichuris from macaques (TMF31) and that from baboon (TPM1) were genetically similar, with a difference in nucleotide and amino acid sequence of 0.25% and 0.41%, and having 14,091 and 14,098 bp, respectively, and both genomes being similar with that of T. trichiura from humans from Uganda, which was 14,079 bp in length, and with TTB1 (13,984 bp in length) from Trichuris sp. from P. hamadryas [19], with a sequence variation of 0.28–0.47%. In contrast, the complete mt genome of the other haplotype from macaques (TMM5) was 14,047 bp, like human T. trichiura from China that was 14,046 bp in length [3], showing a nucleotide difference of 15.9%. Moreover, the mt genome of Trichuris sp. from P. anubis (USA) was 14,009 bp [19], with a nucleotide difference to TMM5 of 15.8%. A substantial level of nucleotide difference was detected between TMF31 (Clade 3) with respect to Trichuris sp. included in clades 1 and 2 (15.8–18.8%). Comparison of the nucleotide and amino acid sequences between TMF31 and TMM5 was 18.7% and 14.5%, respectively. The sequence variation detected in the 13 PCGs between TMF31 and TMM5 was 20.3% (nucleotide sequence) and 34.4% (amino acid sequence). The percentage of dissimilarity observed between the different sequences corresponded to previously cited ranges within different nematode species. Thus, Hawash et al. [19] suggested the presence of two Trichuris species with a difference in nucleotide and amino acid sequences of around 18.8% and 14.6%, respectively, of Trichuris from Ugandan humans and Chinese humans. Furthermore, T. suis and T. trichiura presented an amino acid difference of 33.3–39.2% [19], T. trichiura from human and Trichuris sp. from leaf monkey was 29.4% [17], T. ovis and T. discolor ranged from 11.0–33.9% [16], 11.7% between Wucheria bancrofti and B. malayi [42], 10.3% between Chabertia ovina and C. erschowi [43], 4–18% between different Trichinella spp. [18] and 4.12% between and [44]. Blouin [45] indicated that the difference in mt genome sequences between closely related species was normally 10–20%. Thus, the molecular evidence presented in the present manuscript supports the hypothesis that the haplotypes TMF31 and TMM5 of Trichuris from M. sylvanus display differences in amino acid and nucleotide sequences that are within the range of those previously reported by different authors to consider them as different species. On the other hand, the complete mt genomes of Trichuris spp. from different hosts were evaluated to clarify the evolutionary relationships. Considering that complete mt Life 2021, 11, 126 12 of 15

genome sequences are maternally inherited and mutate at a rapid rate related to nuclear genes, theses markers could prove useful for evolutionary and phylogenetic analyses [46]. Phylogenetics trees inferred from both datasets (sequences dataset of 12 PCGs among enoplid nematodes and nucleotide sequence datasets with the 13 PCGs and the two rRNAs (rrnL and rrnS)) showed congruence with a high support for the differentiation between Trichuris spp. and the different clades within Trichuris populations parasitizing humans and NHPs (clade 1, clade 2 and clade 3). Furthermore, the monophyly of the genus Trichuris was strongly supported and grouping these species of Trichuris (traditionally named as ) with T. pseuospirallis (Trichinellida,) with the exclusion of the members of the Dorylaimida and Mermithida (Figure4). Similar results were reported by Liu et al. [3,16] who characterized the complete mitochondrial genomes of several whipworms and compared them with other enoplid nematodes. The atp8 gene is present in mitogenomes of the Order Trichinellida (Trichuris and Trichinella species), but it is usually absent in mitogenomes of members of the phylum Nematoda [3,13,15–19]. These authors reported the lack of atp8 in the mtDNA of Chro- madorean nematodes (traditionally named as Class ) and it could be derived, within that lineage, after the divergence of Chromadorean nematodes from other groups. Furthermore, phylogenetic inferences confirm the clear relationships between Trichinellida and Mermithida correlating both groups with animal parasites of nematodes separated of Dorylaimida, corresponding to free-living nematodes and plant parasitic nematodes [47]. The phylogenetic study also supported the evolution of the different Trichuris species showing T. trichiura (clade 3), to be closer to Trichuris spp. (clade 1 and clade 2) parasitizing humans and NHPs. As reported by previous authors [19,23,38,39,41,48], Trichuris popula- tions parasitizing humans and NHPs showed a species complex in primates. In addition, the high difference in nucleotide and amino acid sequences between T. trichiura (clade 3) and the minority population of Trichuris sp. from M. sylvanus (clade 2) and Trichuris sp. from humans and NHPs from China, Japan, and USA (clade 1) was confirmed in the phylogenetic trees with strong support for the different clades. Thus, based on our results, we suggest the existence of two cryptic species parasitizing M. sylvanus. Trichuris genus is a likely candidate to containing cryptic species as it has a wide geographical distribution and infects several host species [49]. As revealed by recent studies, there is more than one taxon capable of infecting humans and other primates, including individuals in captivity, suggesting that T. trichiura should be considered a complex species that includes different cryptic units [19]. In addition, and based on morphobiometric and molecular parameters, new species of Trichuris have been described in primates, such as T. rhinopiptheroxella [20], that was found in the golden snub-nosed monkey (Rhinopithecus roxellana), Trichuris colobae from Colobus guereza kikuyensis [50], and Trichuris ursinus from Papio ursinus [51]. As a result, it has been confirmed that T. trichiura and other Trichuris species are present in humans and NHPs. This fact has two main epidemiological repercussions—(i) the zoonotic potential of Trichuris spp. of NHPs for humans. This is particularly important when humans and NHPs are living in proximity, as is becoming increasingly common with human encroachment into habitats where NHPs accessing gardens and farms in search of food and has significant implications for both human health and wildlife conservation [52]. This implies that in communities where access to NHPs is common, simple public health measures should be encouraged, including thorough handwashing with soap (particularly for children) and rising and cooking of vegetables, and (ii) the presence of different cryptic species might also be very important for implementation of appropriate control strategies. Different control strategies for Opistorchirs viverrini have been identified due to the existence of different cryptic species based on the different fecundity as measured by eggs/g/worm [53].

5. Conclusions In the present study, based on complete mt genome analyses, the molecular data suggested two distinct species in whipworms isolated from M. sylvanus. The whipworms Life 2021, 11, 126 13 of 15

from P. papio, TPM1, and TMF31 from M. sylvanus are T. trichiura since the sequences were within the majority clade, with sequences from Ugandan humans and P. hamadryas (Europe)-derived Trichuris. Further, we suggested the existence of two cryptic species parasitizing M. sylvanus. Moreover, a major source of mitochondrial markers is given and may be used as the basis of subsequent epidemiological research, to clarify appropriate control measures and transmission routes.

Supplementary Materials: The following are available online at https://www.mdpi.com/2075-1 729/11/2/126/s1, Table S1: PCR mix, primers and conditions used for each molecular marker sequenced in the present study, Table S2: Pairwise nucleotide and amino acid distances for the different 13 PCGs and RNAs (rrnS and rrnL) genes for Trichuris species from different primates and human hosts. Nucleotide genetics distances are given below the diagonal and amino acid genetic distances above the diagonal, Table S3: Nucleotide composition (%) of the mt genomes studied. Author Contributions: Conceptualization, C.C. and R.C.; methodology, J.R. and R.C.; software, J.R.; validation, C.C., J.R. and R.C.; formal analyses, J.R. and R.C.; investigation, C.C., J.R. and C.R.; writing—original draft preparation, C.C., J.R. and R.C.; writing—review and editing, C.C., J.R. and R.C.; supervision, C.C.; project administration, C.C.; funding acquisition, C.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was financially supported by a grant from the Ministry of Economy, Industry and Competitiveness (CGL2017-83057), which included FEDER/Ministry of Science, Innovation and Universities – State Research Agency/ CGL2017-83057, the Junta de Andalucía (BIO-338) and a grant from the V and VI Plan Propio de Investigación of the University of Seville, Spain. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are openly available in GenBank database (GenBank accession nos. MW448470-2). Acknowledgments: We thank Ruiz de la Haba for his technical scientific advice, and Zoo Castellar (Cádiz, Spain) for providing samples of Trichuris spp. from M. sylvanus which naturally died and Parque Natural de Cabárceno (Cantabria, Spain) for providing samples of T. trichiura from P. papio. Conflicts of Interest: The authors declare no conflict of interest.

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